研究者業績
基本情報
- 所属
- 千葉大学 大学院理学研究院生物学研究部門分子細胞生物学講座 教授 (大学院理学研究院長)
- 学位
- 博士（理学）(1993年3月 東京大学)
- J-GLOBAL ID
- 201801014413462567
- researchmap会員ID
- B000305553
経歴
4-
2006年4月 - 現在
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2000年1月 - 2006年3月
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1994年12月 - 1999年12月
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1993年4月 - 1994年11月
学歴
2-
1988年4月 - 1993年3月
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1984年4月 - 1988年3月
委員歴
10-
2023年4月 - 現在
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2020年6月 - 現在
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2019年7月 - 現在
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2015年8月 - 現在
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2017年4月 - 2022年3月
論文
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Genes to cells : devoted to molecular & cellular mechanisms 2023年9月12日 査読有りTor complex 1 (TORC1), a master regulator of cell growth, is an evolutionarily conserved protein kinase within eukaryotic organisms. To control cell growth, TORC1 governs translational processes by phosphorylating its substrate proteins in response to cellular nutritional cues. Mammalian TORC1 (mTORC1) assumes the responsibility of phosphorylating the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) to regulate its interaction with eIF4E. The budding yeast Saccharomyces cerevisiae possesses a pair of 4E-BP genes, CAF20 and EAP1. However, the extent to which the TORC1-4E-BP axis regulates translational initiation in yeast remains uncertain. In this study, we demonstrated the influence of TORC1 on the phosphorylation status of Caf20 in vivo, as well as the direct phosphorylation of Caf20 by TORC1 in vitro. Furthermore, we found the TORC1-dependent recruitment of Caf20 to the 80S ribosome. Consequently, our study proposes a plausible involvement of yeast's 4E-BP in the efficacy of translation initiation, an aspect under the control of TORC1.
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Autophagy Reports 2(1) 2023年4月6日 査読有り
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Scientific Reports 2023年3月28日
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STAR Protocols 2023年3月 査読有り最終著者責任著者
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Molecular Biology of the Cell 2023年2月3日<jats:p> The endoplasmic reticulum (ER) is a major cell compartment where protein synthesis, folding and post-translational modifications occur with assistance from a wide variety of chaperones and enzymes. Quality control systems selectively eliminate abnormal proteins that accumulate inside the ER due to cellular stresses. ER-phagy, i.e., selective autophagy of the ER, is a mechanism that maintains or re-establishes cellular and ER-specific homeostasis through removal of abnormal proteins. However, how ER luminal proteins are recognized by the ER-phagy machinery remains unclear. Here, we applied the aggregation-prone protein, six-repeated islet amyloid polypeptide (6xIAPP), as a model ER-phagy substrate, and found that cell cycle progression 1 (CCPG1), which is an ER-phagy receptor, efficiently mediates its degradation via ER-phagy. We also identified prolyl 3-hydroxylase family member 4 (P3H4) as an endogenous cargo of CCPG1-dependent ER-phagy. The ER luminal region of CCPG1 contains several highly conserved regions that we refer to as cargo interaction regions (CIRs); these directly interact with specific luminal cargos for ER-phagy. Notably, 6xIAPP and P3H4 directly interact with different CIRs. These findings indicate that CCPG1 is a bispecific ER-phagy receptor for ER luminal proteins and the autophagosomal membrane that contributes to the efficient removal of aberrant ER-resident proteins through ER-phagy. </jats:p>
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Antioxidants 11(12) 2373-2373 2022年11月30日 査読有りGentirigeoside B (GTS B) is a dammaren-type triterpenoid glycoside isolated from G. rigescens Franch, a traditional Chinese medicinal plant. In the present study, the evaluation of the anti-aging effect and action mechanism analysis for this compound were conducted. GTS B significantly extended the replicative lifespan and chronological lifespan of yeast at doses of 1, 3 and 10 μM. Furthermore, the inhibition of Sch9 and activity increase of Rim15, Msn2 proteins which located downstream of TORC1 signaling pathway were observed after treatment with GTS B. Additionally, autophagy of yeast was increased. In addition, GTS B significantly improved survival rate of yeast under oxidative stress conditions as well as reduced the levels of ROS and MDA. It also increased the gene expression and enzymatic activities of key anti-oxidative enzymes such as Sod1, Sod2, Cat and Gpx. However, this molecule failed to extend the lifespan of yeast mutants such as ∆cat, ∆gpx, ∆sod1, ∆sod2, ∆skn7 and ∆uth1. These results suggested that GTS B exerts an anti-aging effect via inhibition of the TORC1/Sch9/Rim15/Msn signaling pathway and enhancement of autophagy. Therefore, GTS B may be a promising candidate molecule to develop leading compounds for the treatment of aging and age-related disorders.
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Photonics 9(9) 602-602 2022年8月25日 査読有りWe report on the development of an ultrawideband and high-resolution THz spectroscopic system, which operates over a frequency range of 2–13.5 THz. This work goes beyond conventional THz spectroscopic measurements and demonstrates the capacity to differentiate D- and L-glucoses, which have the same molecular conformation and crystal structure. Furthermore, this system facilitates the structural identification of glucose microcrystals, which exists in α- and β-forms, revealing that L-glucose microcrystals are formed of a mixture of α- and β-D-glucose with approximately equal weight.
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iScience 2022年8月 査読有り最終著者責任著者
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Carcinogenesis 43(7) 693-703 2022年4月8日 査読有りEpidemiological studies have revealed a radiation-related increase in the risk of developing acute lymphoblastic leukemia (ALL). Our recent study revealed early induction and increased risk of precursor B-cell (pB) lymphomas in mice after radiation exposure. However, the genomic landscape of radiation-induced B-cell lymphomas remains unclear. To identify the relevant genetic alterations in mice, whole-exome sequencing was performed on both early-onset and late-onset B-cell lymphomas that developed spontaneously or after gamma-irradiation. In addition to multiple driver mutations, the data revealed that interstitial deletion of chromosome 4, including Pax5, and missense mutations in Jak3 are unique genomic alterations in radiation-induced, early-onset B-cell lymphomas. RNA sequencing revealed a pB-cell-type gene-expression profile with no involvement of known fusion genes for human ALLs in the early-onset B-cell lymphomas. Activation of Jak3/Stat5 signaling in early-onset B-cell lymphomas was validated using western capillary electrophoresis. Those features were similar to those of Philadelphia chromosome-like ALL. Our data suggest a critical role for Pax5 loss-of-function mutations in initiating B-cell leukemogenesis coupled with activation of Jak3/Stat5 signaling as a basis for the rapid development of radiation-induced pB-ALL. These molecular signatures for radiation-induced cancers will inform both risk assessment and potential targeted therapies for pB-ALL.
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Cell death discovery 8(1) 37-37 2022年1月25日 査読有りAutophagy plays important role in the intracellular protein quality control system by degrading abnormal organelles and proteins, including large protein complexes such as ribosomes. The eukaryotic chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC), also called chaperonin-containing TCP1 (CCT), is a 1-MDa hetero-oligomer complex comprising 16 subunits that facilitates the folding of ~10% of the cellular proteome that contains actin. However, the quality control mechanism of TRiC remains unclear. To monitor the autophagic degradation of TRiC, we generated TCP1α-RFP-GFP knock-in HeLa cells using a CRISPR/Cas9-knock-in system with an RFP-GFP donor vector. We analyzed the autophagic degradation of TRiC under several stress conditions and found that treatment with actin (de)polymerization inhibitors increased the lysosomal degradation of TRiC, which was localized in lysosomes and suppressed by deficiency of autophagy-related genes. Furthermore, we found that treatment with actin (de)polymerization inhibitors increased the association between TRiC and unfolded actin, suggesting that TRiC was inactivated. Moreover, unfolded actin mutants were degraded by autophagy. Taken together, our results indicate that autophagy eliminates inactivated TRiC, serving as a quality control system.
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Antioxidants 11(2) 214-214 2022年1月23日 査読有り
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STAR Protocols 2(4) 100975-100975 2021年12月 査読有りEndocytic internalization of extracellular proteins plays roles in signaling, nutrient uptake, immunity, and extracellular protein quality control. However, there are few protocols for analyzing the lysosomal degradation of extracellular protein. Here, we purified secreted proteins fused with pH-sensitive GFP and acid- and protease-resistant RFP from mammalian cells and describe an internalization assay for mammalian cells. This protocol enables quantification of cellular uptake and lysosomal degradation of protein-of-interest (POI) via cell biological and biochemical analyses. For full details on the use and execution of this protocol, please refer to Itakura et al. (2020).
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The Journal of biological chemistry 297(5) 101279-101279 2021年10月5日 査読有りMitochondria are essential organelles that carry out a number of pivotal metabolic processes and maintain cellular homeostasis. Mitochondrial dysfunction caused by various stresses is associated with many diseases such as type 2 diabetes, obesity, cancer, heart failure, neurodegenerative disorders, and aging. Therefore, it is important to understand the stimuli that induce mitochondrial stress. However, broad analysis of mitochondrial stress has not been carried out to date. Here, we present a set of fluorescent tools, called mito-Pain (mitochondrial PINK1 accumulation index), which enable the labeling of stressed mitochondria. Mito-Pain uses PTEN-induced putative kinase 1 (PINK1) stabilization on mitochondria and quantifies mitochondrial stress levels by comparison with PINK1-GFP, which is stabilized under mitochondrial stress, and RFP-Omp25, which is constitutively localized on mitochondria. To identify compounds that induce mitochondrial stress, we screened a library of 3374 compounds using mito-Pain and identified 57 compounds as mitochondrial stress inducers. Furthermore, we classified each compound into several categories based on mitochondrial response: depolarization, mitochondrial morphology, or Parkin recruitment. Parkin recruitment to mitochondria was often associated with mitochondrial depolarization and aggregation, suggesting that Parkin is recruited to heavily damaged mitochondria. In addition, many of the compounds led to various mitochondrial morphological changes, including fragmentation, aggregation, elongation, and swelling, with or without Parkin recruitment or mitochondrial depolarization. We also found that several compounds induced an ectopic response of Parkin, leading to the formation of cytosolic puncta dependent on PINK1. Thus, mito-Pain enables the detection of stressed mitochondria under a wide variety of conditions and provides insights into mitochondrial quality control systems.
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Antioxidants (Basel, Switzerland) 10(6) 987-987 2021年6月21日 査読有りTwo compounds that can prolong the replicative lifespan of yeast, geniposidic acid (Compound 1) and geniposide (Compound 2), were isolated from Gardenia jasminoides Ellis. Compared with Compound 1, Compound 2 was different at C11 and showed better bioactivity. On this basis, seven new geniposidic derivatives (3–9) were synthesized. Geniposidic 4-isoamyl ester (8, GENI), which remarkably prolonged the replicative and chronological lifespans of K6001 yeast at 1 µM, was used as the lead compound. Autophagy and antioxidative stress were examined to clarify the antiaging mechanism of GENI. GENI increased the enzymes activities and gene expression levels of superoxide dismutase (SOD) and reduced the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) to improve the survival rate of yeast under oxidative stress. In addition, GENI did not extend the replicative lifespan of ∆sod1, ∆sod2, ∆uth1, ∆skn7, ∆cat, and ∆gpx mutants with K6001 background. The free green fluorescent protein (GFP) signal from the cleavage of GFP-Atg8 was increased by GENI. The protein level of free GFP showed a considerable increase and was time-dependent. Furthermore, GENI failed to extend the replicative lifespans of ∆atg32 and ∆atg2 yeast mutants. These results indicated that antioxidative stress and autophagy induction were involved in the antiaging effect of GENI.
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Oxidative Medicine and Cellular Longevity 2021 5469849 2021年1月13日 査読有り
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Journal of Radiation Research 61(5) 648-656 2020年9月8日 査読有り<title>Abstract</title> Epidemiological studies of atomic-bomb survivors have revealed an increased risk of lymphoid neoplasm (i.e. acute lymphoblastic leukemia) associated with radiation exposure. In particular, children are more susceptible to radiation-induced precursor lymphoid neoplasm than adults. Although ~75% of human lymphoid tumors are B-cell neoplasms, the carcinogenic risk associated with each stage of differentiation of B-cells after radiation exposure is poorly understood. Therefore, we irradiated mice at infancy or in young adulthood to investigate the effect of age at exposure on the risk of developing B-cell neoplasms. Histopathology was used to confirm the presence of lymphoid neoplasms, and the population of B-cell neoplasms was classified into the precursor B-cell (pro-B and pre-B cell) type and mature B-cell type, according to immunophenotype. The data revealed that precursor B-cell neoplasms were induced soon after radiation exposure in infancy or young adulthood, resulting in a greater risk of developing the neoplasms. This was particularly the case for the pro-B cell type after young adult exposure. Our findings suggest that exposure to radiation at young age increases the risk of developing precursor B-cell neoplasms in humans.
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Oxidative Medicine and Cellular Longevity 2020 1-12 2020年8月3日 査読有りGentiopicroside (GPS), an antiaging secoiridoid glycoside, was isolated from <italic>Gentiana rigescens</italic> Franch, a traditional Chinese medicine. It prolonged the replicative and chronological lifespans of yeast. Autophagy, especially mitophagy, and antioxidative stress were examined to clarify the mechanism of action of this compound. The free green fluorescent protein (GFP) signal from the cleavage of GFP-Atg8 and the colocation signal of MitoTracker Red CMXRos and GFP were increased upon the treatment of GPS. The free GFP in the cytoplasm and free GFP and ubiquitin of mitochondria were significantly increased at the protein levels in the GPS-treated group. GPS increased the expression of an essential autophagy gene, <italic>ATG32</italic> gene, but failed to extend the replicative and chronological lifespans of <italic>ATG32</italic> yeast mutants. GPS increased the survival rate of yeast under oxidative stress condition; enhanced the activities of catalase, superoxide dismutase, and glutathione peroxidase; and decreased the levels of reactive oxygen species and malondialdehyde. The replicative lifespans of <italic>Δsod1</italic>, <italic>Δsod2</italic>, <italic>Δuth1</italic>, and <italic>Δskn7</italic> were not affected by GPS. These results indicated that autophagy, especially mitophagy, and antioxidative stress are involved in the antiaging effect of GPS.
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The Journal of Cell Biology 219(3) 2020年3月2日 査読有りThe accumulation of aberrant proteins leads to various neurodegenerative disorders. Mammalian cells contain several intracellular protein degradation systems, including autophagy and proteasomal systems, that selectively remove aberrant intracellular proteins. Although mammals contain not only intracellular but also extracellular proteins, the mechanism underlying the quality control of aberrant extracellular proteins is poorly understood. Here, using a novel quantitative fluorescence assay and genome-wide CRISPR screening, we identified the receptor-mediated degradation pathway by which misfolded extracellular proteins are selectively captured by the extracellular chaperone Clusterin and undergo endocytosis via the cell surface heparan sulfate (HS) receptor. Biochemical analyses revealed that positively charged residues on Clusterin electrostatically interact with negatively charged HS. Furthermore, the Clusterin-HS pathway facilitates the degradation of amyloid β peptide and diverse leaked cytosolic proteins in extracellular space. Our results identify a novel protein quality control system for preserving extracellular proteostasis and highlight its role in preventing diseases associated with aberrant extracellular proteins.
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Cellular Signalling 62 109344-109344 2019年10月 査読有り
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Scientific reports 9(1) 11635-11635 2019年8月12日 査読有りLysosomes are largely responsible for significant degradation of intracellular and extracellular proteins via the secretory pathway, autophagy, and endocytosis. Therefore, dysregulation of lysosomal homeostasis influences diverse cellular functions. However, a straightforward and quantitative method to measure the integrity of the lysosomal pathway has not been developed. Here, we report the plasmid-based lysosomal-METRIQ (MEasurement of protein Transporting integrity by RatIo Quantification) probe that enables simple quantification of lysosomal integrity by lysosomal green and cytosolic red fluorescent proteins using a flow cytometer. In cultured cells, the lysosomal-METRIQ probe detected not only suppression of the lysosomal pathway but also upregulation of lysosomal activity such as lysosomal biogenesis. To identify factors involved in lysosomal homeostasis, we carried out compound screening and found that the cyclin-dependent kinase (CDK) inhibitors kenpaullone and purvalanol A induce synthesis of cathepsin D and an increase in the number of lysosomes. Subsequent studies revealed that CDK5 maintains lysosomal homeostasis independently of cell cycle arrest. Our results suggest that the lysosomal-METRIQ probe is an effective and efficient tool for measuring lysosomal activity in mammalian cells.
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Biochemical and Biophysical Research Communications 516(1) 133-137 2019年8月 査読有り
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Genes to Cells 24(8) 546-558 2019年8月 査読有り
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Oxidative Medicine and Cellular Longevity 2019年6月2日 査読有り
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Communicative & Integrative Biology 11(2) 1-4 2018年3月4日 査読有り
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Development (Cambridge, England) 145(4) 2018年2月23日 査読有りAlthough autophagy is classically viewed as a non-selective degradation system, recent studies have revealed that various forms of selective autophagy also play crucial physiological roles. However, the induction of selective autophagy is not well understood. In this study, we established a forced selective autophagy system using a fusion of an autophagy adaptor and a substrate-binding protein. In both mammalian cells and fertilized mouse embryos, efficient forced lipophagy was induced by expression of a fusion of p62 (Sqstm1) and a lipid droplet (LD)-binding domain. In mouse embryos, induction of forced lipophagy caused a reduction in LD size and number, and decreased the triglyceride level throughout embryonic development, resulting in developmental retardation. Furthermore, lipophagy-induced embryos could eliminate excess LDs and were tolerant of lipotoxicity. Thus, by inducing forced lipophagy, expression of the p62 fusion protein generated LD-depleted cells, revealing an unexpected role of LD during preimplantation development.
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Molecular biology of the cell 29(4) 510-522 2018年2月15日 査読有りTarget of rapamycin complex 1 (TORC1) is a central cellular signaling coordinator that allows eukaryotic cells to adapt to the environment. In the budding yeast, Saccharomyces cerevisiae, TORC1 senses nitrogen and various stressors and modulates proteosynthesis, nitrogen uptake and metabolism, stress responses, and autophagy. There is some indication that TORC1 may regulate these downstream pathways individually. However, the potential mechanisms for such differential regulation are unknown. Here we show that the serine/threonine protein kinase Sch9 branch of TORC1 signaling depends specifically on the integrity of the vacuolar membrane, and this dependency originates in changes in Sch9 localization reflected by phosphatidylinositol 3,5-bisphosphate. Moreover, oxidative stress induces the delocalization of Sch9 from vacuoles, contributing to the persistent inhibition of the Sch9 branch after stress. Thus, our results establish that regulation of the vacuolar localization of Sch9 serves as a selective switch for the Sch9 branch in divergent TORC1 signaling. We propose that the Sch9 branch integrates the intrinsic activity of TORC1 kinase and vacuolar status, which is monitored by the phospholipids of the vacuolar membrane, into the regulation of macromolecular synthesis.
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Biochemical and Biophysical Research Communications 496(3) 834-839 2018年2月12日 査読有り
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FEBS letters 591(9) 1199-1211 2017年5月 査読有りBasal autophagy plays an essential role as a protein quality control system. Although it has been demonstrated that the loss of autophagy results in the accumulation of ubiquitin-positive aggregates and the development of neurodegenerative diseases, the precise autophagy substrate(s) remain unclear. Here, we determined whether ubiquitinated proteins are direct substrates for basal autophagy using a fluorescent ratiometric probe for ubiquitin. We show that the degradation of polyubiquitinated proteins is not dependent on basal autophagy. Although ubiquitin-positive aggregates are observed in autophagy knockout cultured cells, the aggregates consist of soluble and mobile polyubiquitinated proteins, which are trapped by p62 without an increase in the total amount of ubiquitinated proteins. These results suggest that ubiquitinated proteins are not major targets for basal autophagy.
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JOURNAL OF BIOCHEMISTRY 161(3) 297-308 2017年3月 査読有りPex19p harbouring a prenylation CAAX box functions as a chaperone and transporter for peroxisomal membrane proteins in membrane assembly. By functional phenotype-complementation assay using a pex19 Chinese hamster ovary cell mutant ZP119, we herein cloned a rat cDNA encoding a protein similar to Pex19p, but with a C-terminal hydrophobic segment in place of the CAAX box region. The transcript of this gene was highly induced by treatment of rats with a peroxisome proliferator, clofibrate, hence termed PEX19i, while the other three less prominently inducible PEX19 variants encoded authentic Pex19p but differed in the length of 3' non-coding region. Pex19pi restored peroxisomes in ZP119 with slightly lower efficiency than Pex19p, showing apparently weaker interaction with Pex11p beta essential for peroxisome proliferation. However, the C-terminal region of Pex19p was not essential for the association of Pex19p with peroxisomal membrane and interaction with membrane assembly factors, Pex3p and Pex16p. Non-prenylated Pex19p interacted with a membrane protein cargo, Pex14p, but more weakly than Pex19pi and the farnesylated Pex19p. Thus, PEX19i most likely plays important roles involving the membrane formation at early stages, in prompt response to peroxisome proliferation. Similar types of PEX19 mRNA variants were also elevated in mouse regenerating liver.
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PLOS ONE 11(3) 2016年3月 査読有りThe chronological lifespan of Saccharomyces cerevisiae represents the duration of cell survival in the postdiauxic and stationary phases. Using a prototrophic strain derived from the standard auxotrophic laboratory strain BY4742, we showed that supplementation of nonessential amino acids to a synthetic defined (SD) medium increases maximal cell growth and extends the chronological lifespan. The positive effects of amino acids can be reproduced by modulating the medium pH, indicating that amino acids contribute to chronological longevity in a cell-extrinsic manner by alleviating medium acidification. In addition, we showed that the amino acid-mediated effects on extension of chronological longevity are independent of those achieved through a reduction in the TORC1 pathway, which is mediated in a cell-intrinsic manner. Since previous studies showed that extracellular acidification causes mitochondrial dysfunction and leads to cell death, our results provide a path to premature chronological aging caused by differences in available nitrogen sources. Moreover, acidification of culture medium is generally associated with culture duration and cell density; thus, further studies are required on cell physiology of auxotrophic yeast strains during the stationary phase because an insufficient supply of essential amino acids may cause alterations in environmental conditions.
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Autophagy 12(1) 1-222 2016年 査読有り
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OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2016 1 2016年 査読有りParishin is a phenolic glucoside isolated from Gastrodia elata, which is an important traditional Chinese medicine; this glucoside significantly extended the replicative lifespan of K6001 yeast at 3, 10, and 30 mu M. To clarify its mechanism of action, assessment of oxidative stress resistance, superoxide dismutase (SOD) activity, malondialdehyde (MDA), and reactive oxygen species (ROS) assays, replicative lifespans of sod1, sod2, uth1, and skn7 yeast mutants, and real-time quantitative PCR (RT-PCR) analysis were conducted. The significant increase of cell survival rate in oxidative stress condition was observed in parishin-treated groups. Silent information regulator 2 (Sir2) gene expression and SOD activity were significantly increased after treating parishin in normal condition. Meanwhile, the levels of ROS and MDA in yeast were significantly decreased. The replicative lifespans of sod1, sod2, uth1, and skn7 mutants of K6001 yeast were not affected by parishin. We also found that parishin could decrease the gene expression of TORC1, ribosomal protein S26A (RPS26A), and ribosomal protein L9A (RPL9A) in the target of rapamycin (TOR) signaling pathway. Gene expression levels of RPS26A and RPL9A in uth1, as well as in uth1, sir2 double mutants, were significantly lower than those of the control group. Besides, TORC1 gene expression in uth1 mutant of K6001 yeast was inhibited significantly. These results suggested that parishin exhibited antiaging effects via regulation of Sir2/Uth1/TOR signaling pathway.
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MOLECULAR BIOLOGY OF THE CELL 27(2) 382-396 2016年1月 査読有りTORC1 regulates cellular growth, metabolism, and autophagy by integrating various signals, including nutrient availability, through the small GTPases RagA/B/C/D in mammals and Gtr1/2 in budding yeast. Rag/Gtr is anchored to the lysosomal/vacuolar membrane by the scaffold protein complex Ragulator/Ego. Here we show that Ego consists of Ego1 and Ego3, and novel subunit Ego2. The Delta ego2 mutant exhibited only partial defects both in Gtr1-dependent TORC1 activation and Gtr1 localization on the vacuole. Ego1/2/3, Gtr1/2, and Tor1/Tco89 were colocalized on the vacuole and associated puncta. When Gtr1 was in its GTP-bound form and TORC1 was active, these proteins were preferentially localized on the vacuolar membrane, whereas when Gtr1 was in its GDP-bound form, they were mostly localized on the puncta. The localization of TORC1 to puncta was further facilitated by direct binding to Gtr2, which is involved in suppression of TORC1 activity. Thus regulation of TORC1 activity through Gtr1/Gtr2 is tightly coupled to the dynamic relocation of these proteins.
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NUCLEIC ACIDS RESEARCH 41(14) 6930-6941 2013年8月 査読有りWhen DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway.
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INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 14(3) 4461-4475 2013年3月 査読有りNolinospiroside F is a steroidal saponin isolated from Ophiopogon japonicus (O. japonicus). In this study, we found that nolinospiroside F significantly extends the replicative lifespan of K6001 yeast at doses of 1, 3 and 10 mu M, indicating that it has an anti-aging effect. This may be attributed to its anti-oxidative effect, as nolinospiroside F could increase yeast survival under oxidative stress conditions and decrease the level of malondialdehyde (MDA), an oxidative stress biomarker. It could also increase anti-oxidative stress genes, SOD1 and SOD2, expression, and the activity of superoxide dismutase (SOD). It increase the activity of SIRT1, an upstream inducer of SOD2 expression. In sod1 and sod2 mutant yeast strains, nolinospiroside F failed to extend their replicative lifespan. These results indicate that SOD participates in the anti-aging effect of nolinospiroside F. Furthermore, nolinospiroside F inhibited the expression of UTH1, a yeast-aging gene that is involved in the oxidative stress of yeast, and failed to extend the replicative lifespan of uth1 or skn7 mutant yeast cells. SKN7 is the transcriptional activator of UTH1. We also demonstrate that SOD and UTH1 regulate each other's expression. Together, these results suggest that SOD and UTH1 genes are required for and play interactive roles in nolinospiroside F-mediated yeast lifespan extension.
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PLOS GENETICS 9(1) 2013年1月 査読有りThe coordination of subcellular processes during adaptation to environmental change is a key feature of biological systems. Starvation of essential nutrients slows cell cycling and ultimately causes G1 arrest, and nitrogen starvation delays G2/M progression. Here, we show that budding yeast cells can be efficiently returned to the G1 phase under starvation conditions in an autophagy-dependent manner. Starvation attenuates TORC1 activity, causing a G2/M delay in a Swe1-dependent checkpoint mechanism, and starvation-induced autophagy assists in the recovery from a G2/M delay by supplying amino acids required for cell growth. Persistent delay of the cell cycle by a deficiency in autophagy causes aberrant nuclear division without sufficient cell growth, leading to an increased frequency in aneuploidy after refeeding the nitrogen source. Our data establish the role of autophagy in genome stability through modulation of cell division under conditions that repress cell growth.
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BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76(4) 640-645 2012年4月 査読有りThis study used a replicative lifespan assay of K6001 yeast to screen anti-aging food factors in commercial flavonoids. Hesperidin derived from the Citrus genus extended the lifespan of yeast at doses of 5 and 10 mu M as compared with the control group (p < 0.01, p < 0.01). Reactive oxygen species (ROS), real-time PCR (RTPCR), and lifespan assays of uth1 and skn7 mutants with the K6001 background were used to study the anti-aging mechanisms in yeast. The results indicate that hesperidin significantly inhibits the ROS of yeast, and UTH1 gene expression, and that SKN7 gene are involved in hesperidin-mediated lifespan extension. Further, increases in the Sir2 homolog, SIRT1 activity, and SOD gene expression were confirmed at doses of 5 (p < 0.01) and 10 mu m (p < 0.05). This suggests that Sir2, UTH1 genes, and ROS inhibition after administration of hesperidin have important roles in the anti-aging effects of yeast. However, the aglycon hesperetin did not exhibit anti-aging effects in yeast.
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AGING-US 3(11) 1098-1109 2011年11月 査読有りThe O-type forkhead domain transcription factor (FOXO) is involved in many biological processes such as aging, the oxidative stress response, and growth regulation. FOXO activity is tightly controlled within cells. In particular, growth factor signaling pathways and the oxidative stress response can both stimulate nuclear translocation of this transcription factor. Here, we show that tetrahydrocurcumin (THC), a curcumin metabolite, regulates the oxidative stress response and aging via FOXO. In NIH3T3 cells, THC induced nuclear accumulation of FOXO4, a member of the FOXO family of transcription factors, by inhibiting phosphorylation of protein kinase B (PKB)/Akt. In Drosophila melanogaster, THC attenuated the oxidative stress response, an effect that was blocked in a foxo mutant background. THC also extended the life span of Drosophila under normal conditions, and loss of either foxo or Sir2 activity eliminated this effect. Based on these results, THC may regulate the aging process via an evolutionarily conserved signaling pathway that includes both foxo and Sir2.
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DNA Replication-Current Advances 2011年8月 査読有り
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BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75(4) 800-803 2011年4月 査読有りTwo new anti-aging compounds, ganodermasides C and D, were isolated and their structures elucidated. They are novel ergosterols possessing a 4,6,8(14),22-tetraene-3-one unit with unique hydroxylation at C-9. Both of them significantly extended the replicative lifespan of the K6001 yeast strain. Ganodermasides C and D regulated the expression of the gene for UTH1 to prolong the replicative lifespan of yeast.
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BIOORGANIC & MEDICINAL CHEMISTRY 18(3) 999-1002 2010年2月 査読有りTwo novel ergosterol derivatives, ganodermasides A and B, hydroxylated at C-15 were isolated from the methanol extract of spores of a medicinal mushroom, Ganoderma lucidum, showed to extend the replicative life span of Saccharomyces cerevisiae, a yeast of K6001 strain. The stereostructures of ganodermasides A and B were determined based on the spectroscopic analysis and comparison of spectroscopic data. These new sterols have a 4, 6, 8(14), 22-tetraene-3-one unit with a unique hydroxylation at C-15. The anti-aging activity of these compounds on yeast is comparable to a well-known substance, resveratrol. Based on results of the investigation of the mechanism of biological activity, ganodermasides A and B regulated UTH1 expression in order to extend the replicative life span of yeast. (C) 2010 Elsevier Ltd. All rights reserved.
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BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 74(1) 195-198 2010年1月 査読有りDNA replication without telomerase leads to telomere shortening and induces replicative senescence. We round that in a telomerase-deficient budding yeast mutant, the volume of each telomere-shortened cell increased as its growth capacity decreased, and that this process Was associated with changes in vacuolar morphology. Senescence-induced cell expansion required Mec1, a DNA damage-responsive kinase, but not vacuolar SNARE Vam3.
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PLOS ONE 3(5) 2008年5月 査読有りThe target of rapamycin (Tor) protein plays central roles in cell growth. Rapamycin inhibits cell growth and promotes cell cycle arrest at G1 (G0). However, little is known about whether Tor is involved in other stages of the cell division cycle. Here we report that the rapamycin-sensitive Tor complex 1 (TORC1) is involved in G2/M transition in S. cerevisiae. Strains carrying a temperature-sensitive allele of KOG1 (kog1-105) encoding an essential component of TORC1, as well as yeast cell treated with rapamycin show mitotic delay with prolonged G2. Overexpression of Cdc5, the yeast polo-like kinase, rescues the growth defect of kog1-105, and in turn, Cdc5 activity is attenuated in kog1-105 cells. The TORC1-Type2A phosphatase pathway mediates nucleocytoplasmic transport of Cdc5, which is prerequisite for its proper localization and function. The C-terminal polo-box domain of Cdc5 has an inhibitory role in nuclear translocation. Taken together, our results indicate a novel function of Tor in the regulation of cell cycle and proliferation.
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 51(2) 162-168 2006年 査読有り
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MOLECULAR BIOLOGY OF THE CELL 16(12) 5551-5562 2005年12月 査読有りATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.
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SEIKAGAKU 77(3) 233-240 2005年3月 査読有り
MISC
24書籍等出版物
5講演・口頭発表等
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The Second A3 Roundtable meeting on Chemical Probe Research Hub 2017年11月 招待有り
共同研究・競争的資金等の研究課題
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日本学術振興会 科学研究費助成事業 2019年4月 - 2022年3月
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日本学術振興会 科学研究費助成事業 2010年 - 2012年
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日本学術振興会 科学研究費助成事業 2009年 - 2010年
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日本学術振興会 科学研究費助成事業 2006年 - 2009年
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日本学術振興会 科学研究費助成事業 2007年 - 2008年